One step in RNA sequencing (RNA-Seq) is amplification of template RNA against a background of ribosomal RNA. Amplification may involve adapter ligation and since ligation is non-specific, it is desirable to introduce target enrichment by some means. One method takes advantage of the template-switching activity of reverse transcriptase (see for example, U.S. Pat. No. 5,962,272).
One problem of template-switching is that oligonucleotides designed to hybridize with the 3′ end of the reverse transcriptase synthesized first strand cDNA also can form concatemers with each other as the reverse transcriptase switches between templated and non-templated DNA synthesis. This can cause non-specific background and wasted, and therefore costly, sequencing capacity.
Template-switching methods can be applied to the analysis of the transcriptome of single cells to provide gene expression information for use in cell biology studies and disease diagnostics. The initial step in obtaining the transcriptome of a single cell is the isolation of individual cells from a potentially heterogeneous population. This has been described for example, in Saliba, et al., Nucleic Acids Research, 1 doi: 10.1093/nar/gku555 (2014).
Most tissues contain a large number of cell types with distinct lineage, where each cell type contributes to the functioning of the tissue. To understand the relationship among different cell types, single-cell analysis can provide not only genomic information but also transcript information derived from sequencing of mRNA. Studying cells at the single-cell level offers unique opportunities to dissect the interplay between intrinsic cellular processes and extrinsic stimuli such as the local environment or neighboring cells in cell fate determination and can be used to identify cell lineages. Single cell RNA-Seq finds many uses such as, for example, cell lineage analysis; effect of external stimuli on cells, analyzing tissue heterogeneity in cancer biopsies; discovery of rare cells in liquid biopsies; and analyzing nucleic acid content of exosomes.
RNA-Seq has also facilitated the annotation of prokaryotic genomes by defining 5′ and 3′ untranslated regions of mRNAs and discovered many previously unrecognized RNA molecules including an unexpected degree of genome-wide antisense transcription.
The reduction of background and the minimization of unnecessary sequencing would enable RNA-Seq to be more sensitive and cost effective.